Oestrogen-induced apoptosis in colonocytes expressing oestrogen receptor beta

Epidemiological studies of postmenopausal hormone replacement therapy show a reduction in the risk of developing colon cancer, and animal studies using 17beta-oestradiol (E(2)) demonstrate a decreased incidence of chemically-induced colon cancer. Using the colon cancer cell line, COLO205, we found that E(2) induced a dose-dependent increase in DNA fragmentation and nuclear condensation, significant effects being seen at 10(-12 )mol/l. BSA-conjugated E(2), which cannot enter cells, was ineffective at inducing apoptosis in COLO205 cells, indicating that E(2) was not acting through a cell-membrane receptor. E(2) did not induce the morphological changes characteristic of differentiation. Using RT-PCR we found that the oestrogen receptor alpha (ERalpha) isoform was absent in the COLO205 cell line in contrast to CACO-2, LoVo and SW620 cells, but mRNAs for ERbeta1, -beta2, -beta5 and -beta6 isoforms were detected. Western immunoblotting results showed full-length ERbeta protein but no detectable ERalpha in COLO205 cells. In normal human colon tissue samples immunoreactive ERbeta was found but ERalpha was barely detectable. Expression of ERbeta was lost in some colon cancer specimens and reduced in others. We conclude that E(2), through ERbeta, at concentrations found during replacement therapy, may inhibit the development of colon cancer by inducing apoptosis.     

Butyrate and glucose metabolism by colonocytes in experimental colitis in mice

BACKGROUND/AIMS: Impaired colonocyte metabolism of butyrate has been implicated in the aetiopathogenesis of ulcerative colitis. Colonocyte butyrate metabolism was investigated in experimental colitis in mice. METHODS: Colitis was induced in Swiss outbred white mice by oral administration of 4% dextran sulphate sodium (DSS). Colonocytes isolated from colitic and normal control mice were incubated with [(14)C]butyrate or glucose, and production of (14)CO(2), as well as of intermediate metabolites (acetoacetate, beta-hydroxybutyrate and lactate), was measured. The effect of different substrate concentrations on oxidation was also examined. RESULTS: Butyrate oxidation (micromol/h per mg protein; mean (SEM)) was significantly reduced in DSS colitis, values on day 7 of DSS administration being 0.177 (0.007) compared with 0.406 (0.035) for control animals (p<0.001). Glucose oxidation (micromol/h per mg protein; mean (SEM)) on day 7 of DSS administration was significantly higher than in controls (0.06 (0.006) v 0.027 (0.004), p<0.001). Production of beta-hydroxybutyrate was decreased and production of lactate increased in DSS colitis compared with controls. Increasing butyrate concentration from 10 to 80 mM enhanced oxidation in DSS colitis (0.036 (0.002) to 0.285 (0.040), p<0.001), although it continued to remain lower than in controls. Surface and crypt epithelial cells showed similar ratios of butyrate to glucose oxidation. When 1 mM DSS was added to normal colonocytes in vitro, it did not alter butyrate oxidation. The initial histological lesion of DSS administration was very patchy and involved crypt cells. Abnormal butyrate oxidation became apparent only after six days of DSS administration, at which time histological abnormalities were more widespread. CONCLUSIONS: Colonocyte metabolism of butyrate, but not of glucose, is impaired in DSS colitis, and may be important in pathophysiology. Histological abnormalities preceded measurable defects in butyrate oxidation.     

Isolated colonocyte metabolism of glucose, glutamine, n-butyrate, and beta-hydroxybutyrate in malnutrition

The colonic mucosa may be especially vulnerable during starvation and malnutrition, as luminal nutrients make the greatest contribution to its energy production. To investigate possible metabolic changes in the colonic mucosa during nutrient restriction, we studied substrate utilization by colonocytes isolated from three groups of 6-wk-old rats: control, fasted (72 h), and chronically malnourished animals. Isolated colonocytes were incubated with nonlabeled and 14C-labeled substrates (glucose, glutamine, n-butyrate, or beta-hydroxybutyrate). Substrate oxidation and net increase of intermediary metabolites were reduced in fasted and malnourished animals. The effect of fasting on substrate oxidation was greater than that of chronic malnutrition for all substrates tested except n-butyrate. The total ketone body concentrations and beta-hydroxybutyrate to acetoacetate ratios were higher in the fasted and malnourished groups than in controls. The findings suggest that the colonic mucosa responds to nutrient deprivation by a general reduction of oxidative metabolism that is associated with an altered redox state.     

Effect of sulphide on short chain acyl-CoA metabolism in rat colonocytes.

BACKGROUND: It has been proposed that the diminished n-butyrate oxidation observed in ulcerative colitis may be the result of sulphide induced inhibition of short chain acyl-coenzyme A (acyl-CoA) dehydrogenase activity. AIM: To examine the acyl-CoA ester profiles in isolated rat colonic epithelial cells treated in vitro with sodium hydrogen sulphide (NaHS). METHODS: Isolated rat colonic epithelial cell suspensions were incubated for 10 minutes in the presence of [1-14C] n-butyrate (5 mM), with and without NaHS (1.5 mM). Incubations were carried out both in the presence and the absence of exogenous CoA and ATP. Metabolic performance was assessed by 14CO2 production and by acyl-CoA ester production measured by HPLC with ultraviolet detection. RESULTS: Results are given as mean (SEM). For colonocytes incubated in the presence of exogenous CoA and ATP, treatment with NaHS significantly diminished 14CO2 production (control 0.97 (0.06) mumol/g dry weight cells/min, treated 0.26 (0.09) mumol/g dry weight cells/min, p = 0.0019), was associated with an increase in butyryl-CoA concentrations in the final reaction mixture at 10 minutes (control 2.55 (0.28) mumol/g dry weight cells, treated 3.32 (0.32) mumol/g dry weight cells, p = 0.002), and a reduction in crotonyl-CoA concentrations (control 0.274 (0.02) mumol/g dry weight cells, treated 0.120 (0.04) mumol/g dry weight cells, p = 0.008). The mean concentration of acetyl-CoA in the reaction mixture at 10 minutes was not significantly different between control and sulphide treated incubations. There were no significant differences in acyl-CoA ester profiles observed when cells were incubated in the absence of exogenous CoA and ATP. CONCLUSIONS: These results support the view that sulphides inhibit n-butyrate oxidation in colonic epithelial cells by inhibiting short chain acyl dehydrogenation of activated fatty acids.     

Lectin binding to human colonocytes is predictive of colonic neoplasia

OBJECTIVE: The aim of this study was to determine whether lectin binding to exfoliated human colonocytes could be used as a noninvasive test for colorectal polyps or cancer. METHODS: Colonocytes were harvested from 31 patients (10 controls, 10 with adenomatous polyps, and 11 with cancer), incubated with a panel of fluorescent-labeled lectins, and assayed by flow cytometry. RESULTS: The lectins jacalin (JAC) and wheat germ agglutinin (WGA) were useful in predicting the presence of a colorectal neoplasm (p = 0.0018 for JAC and p = 0.0099 for WGA). For JAC, sensitivity reached 81% with a specificity of 80%, and for WGA the sensitivity and specificity were both 75%. CONCLUSIONS: Lectin binding to human colonocytes can predict the presence of malignant and premalignant lesions of the colon, and has potential as a noninvasive screening tool for colorectal neoplasms.     

Substance P mediates antiapoptotic responses in human colonocytes by Akt activation

We examined the hypothesis that substance P (SP) and the neurokinin-1 receptor (NK-1R), both in vitro and in vivo, promote mucosal healing during recovery from colitis by stimulating antiapoptotic pathways in human colonic epithelial cells. For the in vitro experiments, human nontransformed NCM460 colonocytes stably transfected with NK-1R (NCM460-NK-1R cells) were exposed to SP, and cell viability assays, TUNEL assays, and Western blot analyses were used to detect apoptotic and antiapoptotic pathways. SP exposure of NCM460-NK-1R colonocytes stimulated phosphorylation of the antiapoptotic molecule Akt and inhibited tamoxifen-induced cell death and apoptosis evaluated by the cell viability assay and poly(ADP-ribose) polymerase cleavage, respectively. SP-induced phosphorylation of Akt and cleavage of poly(ADP-ribose) polymerase were inhibited by blockade of integrin alphaVbeta3, Jak2, and activation of phosphatidylinositol 3-kinase. For the in vivo experiments, C57BL/6 mice, administered 5% dextran sulfate (DSS) dissolved in tap water for 5 days followed by a 5-day recovery period, were treated with the NK-1R antagonist CJ-12,255 or vehicle. Vehicle-treated mice showed increased colonic Akt phosphorylation and apoptosis compared with mice that received no DSS. In contrast, daily i.p. administration of CJ-12,255 for 5 days post-DSS suppressed Akt activation, exacerbated colitis, and enhanced apoptosis, and pharmacologic inhibition of Akt, either alone or together with CJ-12,255, produced a similar effect. Thus, SP, through NK-1R, possesses antiapoptotic effects in the colonic mucosa by activating Akt, which prevents apoptosis and mediates tissue recovery during colitis.     

Chloride transport in primary cultures of rabbit colonocytes at different stages of development

BACKGROUND & AIMS: Ontogeny of colonic Cl- transport and its regulation has been characterized inadequately. The aim of this report was to study developmental changes in Cl- transport in primary cultures of rabbit distal colonocytes. METHODS: Colonocytes from newborn (7-9 days old), weanling (25-28 days old), and adult (6 months old) rabbits were cultured for 24 hours on a collagen IV matrix, and Cl- transport was measured using the fluoroprobe 6-methoxyquinolyl acetoethyl ester. RESULTS: Cl- permeabilities were dependent on [Cl-]o with maximal rates (in millimoles per liter per second) at [Cl-]o = 75 mmol/L (newborns; 0.15 +/- 0.04; weanlings; 0.2 +/- 0.02; and adults, 0.32 +/- 0.06). Influx was inhibited significantly by the Cl- channel (50 mumol/L diphenylamine-2-carboxylate) and the Na(+)-K(+)- 2Cl- cotransport (10 mumol/L furosemide) inhibitors. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretagogues, prostaglandin E1 (1 mumol/L), forskolin (1 mumol/L), and 8-bromo-cAMP (100 mumol/L), and the protein kinase C activator, phorbol 12-13 dibutyrate (1 mumol/L), increased Cl- influx significantly in all groups with adults showing greatest stimulation. However, taurodeoxycholate (0.025-1 mmol/L) had an effect only in the adult and the guanosine 3',5'-cyclic monophosphate (cGMP) activators STa and 8-bromo-cGMP had no effect. CONCLUSIONS: Rabbit distal colonocytes possess inhibitor-sensitive Cl- permeabilities even in neonates. However, the ontogeny of their regulation depends on the secretagogue-signaling pathway. (Gastroenterology 1996 Dec;111(6):1541-50)     

A phenotypic change of small intestinal epithelium to colonocytes in small intestinal adenomas and adenocarcinomas

OBJECTIVE: Using a novel monoclonal antibody (mAb Das-1) that specifically reacts with colon epithelium, we examined if there is a phenotypic change of small intestinal enterocytes toward colonocytes in small intestinal neoplastic tissue. METHODS: Tissue sections of the small intestine consisting of adenomas (n = 20, five with histories of familial polyposis), adenocarcinomas (eight primary and one metastatic from colon). carcinoids (n = 2), and hyperplastic polyps (n = 3) were examined by a sensitive immunoperoxidase assay using mAb Das-1 (IgM isotype). Normal jejunal (n = 10) and colonic (n = 10) biopsy specimens were also included as additional controls. RESULTS: mAb Das-1 reacted with normal colonic epithelium but not with jejunal mucosa. However, mAb Das-1 reacted strongly with each of the five adenomas (100%) from patients with histories of familial polyposis, but only five of 15 (33%) of the adenomas from nonfamilial polyposis patients, and each of the eight (100%) adenocarcinomas of the small intestine (p < 0.001). The reactivity with the adenomas from nonfamilial polyposis patients was very focal, whereas in the adenomas with familial polyposis the reactivity was more extensive. Each of the eight carcinomas reacted strongly with mAb Das-1. Adjacent normal small intestinal mucosa did not react. Hyperplastic polyps and the carcinoids did not react with mAb Das-1. CONCLUSION: These data demonstrate a phenotypic change in small intestinal epithelium toward the colonic phenotype, particularly in familial polyposis and in adenocarcinomas. mAb Das-1 may be clinically useful in identifying small intestinal adenomas with "high risk" for malignancy, such as in familial polyposis.     

Characterization of the mucins produced by normal human colonocytes in primary culture

Mature goblet cells filled with mucin ready for secretion represent about one third of the cells in primary cultures of human colonocytes. In the present study characterization of the mucins produced by cultured human colonocytes was made by histochemical methods by lectin and monoclonal antibody binding. Two monoclonal antibodies and three lectinsDolichos biflorus (DBA),Helix pomatia (HPA) andArachis hypogea (PNA) recognizing epitopes or sugar haptens characteristic of different stages of mucin glycoprotein maturation, were employed. The reacivity to these probes was tested both on cultured colonocytes and on tissue sections of the normal colon mucosa. The results show that the mucins produced in culture are glycosylated to the mature form, as they show the same reactivity to lectins and antibodies of the mucins expressed in tissue sections of the normal colon mucosa. In addition, it is demonstrated that cultured human colonocytes do not express mucins reactive to PNA, which are characteristic of tumors. Since the cultured colonocytes maintain the expression of differentiated functions for at least three days, they may offer a useful model to study metabolism, function and regulation of colon mucins in health and disease.

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Résumé Des cellules caliciformes matures remplies de mucine prêtes à la sécrétion constituent environ 1/3 de toutes les cellules dans des cultures primaires de colonocytes humains. Au cours de l'étude présente, les mucines produites par des cultures de colonocytes humains ont été typisées par des méthodes histochimiques, par liaison à la lectin et à des anti-corps monoclonaux. Deux anti-corps monoclonaux et trois types de lectin Dolichos biflorus (DBA), Helix pomatio (HPA) et Arachis hypogea (PNA) susceptibles de reconnaïtre les épitopes ou les haptènes d'hydrate de carbone caractéristiques de différentes phases de la maturation des glycoprotéines des mucines ont été emplyés. Les réactivités à ces substances ont été testéees à la fois sur des cultures de colonocytes et sur des segments de tissu muqueux coliques normaux. Les résultats montrent que les mucines produites en culture sont glycosylées dans les formes matures car elles présentent la même réactivité aux lectins et aux anti-corps des mucines tels que mesurés dans des segments de tissue de muqueuse colique normale. De plus, on a démontré que les colonocytes humains cultivés ne produisent pas de mucine réactive au PNA qui sont caractéristiques des tumeurs. Etant donné que les colonocytes humains cultivés conservent l'expression d'une différenciation des fonctions pour au moins trois jours, ils peuvent constituer un modèle utile pour étudier le métabolisme de la fonction et de la régulation des mucines coliques chez le sujet sain et pathologique.

     

Obesity does not increase effects of synthetic ghrelin on human gastric motor functions

BACKGROUND & AIMS: Ghrelin is secreted by the stomach and stimulates food intake. Obese individuals have lower fasting plasma ghrelin levels but increased appetite, suggesting greater responses to endogenous ghrelin in obesity. The aim of this study was to compare effects of exogenous ghrelin (at a dose that stimulates growth hormone [GH] release in the physiologic range) versus placebo on gastric emptying, gastric volume, and postprandial symptoms and determine whether body mass (ranging from normal weight to obesity) influences responses to ghrelin. METHODS: After intravenous bolus synthetic human ghrelin (0.33 mug/kg) or saline, we measured plasma GH, gastric volume, and gastric emptying by combined (99m)Tc-single-photon emission computed tomography and scintigraphy ((111)In egg meal, 300 kcal) and postprandial symptoms using visual analogue scales. RESULTS: In 25 obese subjects (5 men and 20 women; body mass index [BMI], 36 +/- 4 kg/m(2)) and 13 female normal-weight (BMI, 22 +/- 2 kg/m(2)) subjects of similar ages, ghrelin increased GH levels (15.0 +/- 2.4 ng/mL) at 40 minutes postinjection and tended to decrease fasting gastric volumes compared with placebo (P = .059). There were no effects of BMI on treatment response and no differences between ghrelin and saline on postprandial (P = .09) or change in (postprandial minus fasting) gastric volumes, gastric emptying, or aggregate postprandial symptoms. Effects of ghrelin did not differ between obese and normal-weight participants. CONCLUSIONS: At doses that stimulate physiologic GH plasma levels, synthetic ghrelin tended to decrease fasting gastric volumes without altering postprandial volumes or gastric emptying in a predominantly female cohort. The data are not consistent with the hypothesis that higher body mass is associated with increased gastric responsiveness to ghrelin.     

Synergistic efficacy of 3n-butyrate and 5-fluorouracil in human colorectal cancer xenografts via modulation of DNA synthesis

BACKGROUND & AIMS: Butyrate, produced in the colon lumen, maintains mucosal cell homeostasis. Poorly diffusible, its access is compromised in growing colon cancers and absent in distant metastases. Butyrate regulates DNA synthesis. We postulated that systemic administration of butyrate should reduce colon cancer growth and enhance 5-fluorouracil (5-FU) efficacy. METHODS: A stable derivative of butyrate (3n-But) was used. The antitumoral efficacy of 5-FU and 3n-But, alone or combined, was evaluated in human colorectal cancers (hCRCs) subcutaneously, orthotopically, or intrasplenically grafted into nude mice. Thymidylate synthase (TS) and thymidine kinase (TK) mRNA expression, proliferation, apoptosis, and cell cycle alterations were studied. RESULTS: In vivo, 5-FU alone inhibited growth of only 3 of the 12 hCRCs tested and 3n-But alone had no effect; the 5-FU/3n-But combination inhibited growth of all 16 hCRCs tested. The hCRCs differed in their p53 and microsatellite instability status. 5-FU/3n-But decreased TK and TS mRNA expression by 20- and 40-fold, respectively, and TS activity by 75%, stopped cell proliferation without affecting cell differentiation, and significantly enhanced apoptosis. 3n-But potentiated the efficacy of Tomudex and methotrexate, 2 TS inhibitors, but not that of oxaliplatin. In vitro, 5-FU/3n-But inhibited [3H]thymidine but not bromodeoxyuridine incorporation and induced apoptosis in hCRC cell lines. Cells treated with 5-FU/3n-But did not accumulate in G1 nor in S phase of the cell cycle, while 5-FU and 3n-But arrested the cycle in S and in G1 phase, respectively. 3n-But prevented the cell rescue from 5-FU-induced cytotoxicity by uridine or thymidine. CONCLUSIONS: 3n-But and TS inhibitors acted synergistically against colorectal cancers, independently of the genetic alterations of the hCRCs. The mechanism of action of 5-FU/3n-But could be enhanced reduction of TS and prevention of thymidine salvage in DNA synthesis.     

Early functional effects of Clostridium difficile toxin A on human colonocytes

BACKGROUND & AIMS: Previous in vitro studies have shown that Clostridium difficile toxin A is able to directly affect the intestinal epithelial barrier function. The aim of this study was to examine the early effects of toxin A on mucin exocytosis and determine whether this toxin can induce the production of the chemokine interleukin 8 (IL-8) from human colonic epithelial cells. METHODS: Two model systems were used: the HT29-CI.16E colonic goblet cell line and primary cultures of human normal colonocytes. RESULTS: Toxin A exerted a rapid and dose- related inhibition of stimulated mucin exocytosis without altering baseline (constitutive) mucin exocytosis from HT29-CI.16E cells. Toxin A was also able to induce the secretion of IL-8 from both HT29-CI.16E cells and primary cultures of human normal colonocytes, as early as 2-3 hours of incubation. CONCLUSIONS: The results show that while toxin A is able to down-regulate stimulated mucin exocytosis, it is able to up- regulate the secretion of an important chemoattractant chemokine, IL-8. These modifications illustrate the ability of colonocytes to recruit inflammatory and immune cells that will eventually bring about major mucosal damage. (Gastroenterology 1997 Jun;112(6):1887-94)     

The relationship between synthetic and editing functions of the active site of an aminoacyl-tRNA synthetase.

We have analyzed, by site-directed mutagenesis, the molecular basis of the editing function and its relation to the synthetic function of Escherichia coli methionyl-tRNA synthetase. The data obtained fit a model of the active site that partitions an amino acid substrate between synthetic and editing pathways. Hydrophobic and hydrogen bonding interactions direct the cognate substrate methionine through the synthetic pathway and prevent it from entering the editing pathway. Two hydrophobic interactions are proposed: between the side chain of Trp-305 and a methyl group of methionine and between the benzene ring of Tyr-15 and the beta- and gamma-CH2 groups of the substrate. An essential hydrogen bond forms between the OH of Tyr-15 and an electron pair of the sulfur atom of methionine. Consistent with these functions, side chains of Trp-305 and Tyr-15 are localized on opposite sides of the cavity forming a putative methionine binding pocket that is observed in the three-dimensional crystallographic structure of methionyl-tRNA synthetase. Enzymes W305A, Y15A, and Y15F have diminished ability to discriminate against homocysteine in the synthetic reaction, compared to the wild-type enzyme. At the same time, mutant enzymes have lost the ability to discriminate against methionine in the editing reaction and edited Met-AMP to a similar extent as Hcy-AMP. Interactions of residues Arg-233 and Asp-52 of methionyl-tRNA synthetase with the carboxyl and amino groups, respectively, of the substrate, which are essential for the synthetic function, were also essential for the editing function of the enzyme. Deacylation of Met-tRNA to S-methylhomocysteine thiolactone catalyzed by W305A, Y15A, and Y15F mutant enzymes was only slightly impaired relative to the wild-type enzyme. However, enzymes R233Q, R233A, and D52A did not deacylate Met-tRNA. The model also explains why the noncognate homocysteine is edited by methionyl-tRNA synthetase.     

Human colonocytes in primary culture: a model to study epithelial growth,metabolism and differentiation

The purpose of this work was to set up an in vitro model for the study of normal and pathological functions of the colonic epithelium. We have isolated colonic crypts by mild proteolytic digestion and mechanical dissociation of human biopsy material obtained during colonoscopy. The crypts, free of connective tissue, when placed in culture rapidly attached to the substrate and formed colonies containing over 95% of epithelial cells. Histochemical and ultrastructural characterization of the colonies showed the presence of both absorptive and secretory cells, exhibiting a high degree of differentiation. Proliferative activity occurred mostly during the first 24 h and progressively declined thereafter. The cells survived and maintained differential characteristics for at least three days in culture. This method can be used to study normal functions of the colonic epithelium. It may also be employed to investigate both noxious and protective factors in pathological conditions such as inflammatory bowel disease and colorectal neoplasia.

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Résumé Le but de ce travail a été d'établir un modèle in vitro permettant d'étudier les fonctions normales et pathologiques de l'épithélium colique. Nous avons isolé des cryptes coliques par une digestion protéolytique douce et par une dissection mécanique de matériel biopsique prélevé au cours de colonoscopies. Les cryptes dépourvues de tissu conjonctif, placées en milieu de culture, se fixent rapidement sur le substrat et forment des colonies qui renferment plus de 95% de cellules épithéliales. L'histochimie et la caractérisation ultrastructurelle des colonies ont montré la présence de cellules d'absorbtion et de cellules secrétoires présentant un haut degré de différentiation. La prolifération active survient le plus souvent durant les premières 24 heures et diminue progressivement par la suite. Les cellules survivent et maintiennent les caractéristiques de différentiation durant au moins 3 jours en culture. Cette méthode peut être utilisée pour étudier les fonctions normales de l'épithélium colique. Elles peuvent aussi être utilisées pour investiger les facteurs nocifs et protecteurs dans des situations pathologiques telles que les maladies inflammatoires de l'intestin et les tumeurs colorectales.

     

Oxidation of short and medium chain C2-C8 fatty acids in Sprague-Dawley rat colonocytes.

BACKGROUND: The predominant colonic short chain fatty acids, acetate, propionate, and butyrate, are oxidised into CO2 in colonocytes from rat and humans in the preferred order of butyrate (C4) > propionate (C3) > acetate (C2)- hence butyrate is considered to be the principal oxidative substrate for colonocytes. AIMS: To compare colonocyte oxidation of valerate (C5), hexanoate (C6), and octanoate (C8) with that of butyrate. METHODS: Isolated rat colonocytes were incubated in the presence of a concentration range of 1-14C labelled C2-C8 fatty acids. Oxidation rates were obtained by quantifying the production of 14CO2, and Vmax (maximum velocity) and K(m) (Michaelis-Menten constant) were calculated by computer fitting of the data to a Michaelis-Menten plot. RESULTS: The K(m) value of acetate (0.56 (SEM 0.02) mmol/l) was about fourfold higher than the K(m) of butyrate (0.13 (0.01) mmol/l), whereas the K(m) values of valerate (0.19 (0.01) mmol/l), hexanoate (0.19 (0.01) mmol/l), and octanoate (0.16 (0.01) mmol/l) were of the same order of magnitude as the K(m) of butyrate. Acetate did not influence butyrate oxidation, whereas butyrate strongly inhibited the oxidation of acetate. By contrast, valerate, hexanoate, and octanoate inhibited colonocyte oxidation of butyrate equally or more than the reverse inhibitory effect of butyrate on valerate, hexanoate, and octanoate oxidation. The maximum rates of ATP production were in the order of valerate > octanoate = hexanoate > butyrate > acetate (28.47 (0.70), 21.78 (0.75), 21.33 (0.78), 16.12 (0.49), 9.09 (0.34) (mumol/min/g) respectively). CONCLUSIONS: Valerate, hexanoate, and octanoate seem to be excellent substrates for colonocyte oxidation, similar to butyrate. These results may influence the choice of fatty acid composition in enemas used for treatment of patients in whom deficient colonocyte oxidation is suspected-for example, patients with ulcerative colitis and diversion colitis.